Cytotoxic Activity of Aloin

Abstract

In the present study, the cytotoxic activity of aloin, a natural anthraquinone glycoside, was assessed against a breast cancer cell line (T47D) using MTT and clonogenic assays, compared to doxorubicin, an anthracycline analog. The effects of exposure of T47D tumor cells to IC50 values of aloin and doxorubicin (181.5 and 0.17µM, respectively) for 72 h on the mRNA expression levels of a non-receptor tyrosine kinase (JAK-2), a transcription factor (STAT-5a) and some biomarkers for epithelial-mesenchymal transition (EMT) were evaluated.

Exposure to aloin down-regulated the expression levels of JAK-2, STAT-5a and vimentin mRNA in T47D cells, while the expression levels of E-cadherin and ZO-1 mRNA were not significantly changed. On the other hand, exposure of breast tumor cells to doxorubicin up-regulated the expression of E-cadherin mRNA, whereas the expression levels of JAK-2, STAT-5a, ZO-1 and vimentin mRNA were not affected. In conclusion, the chemosensitivity of aloin towards breast tumor cells is due to inhibition of JAK-2/STAT-5a signaling pathway, which results in the inhibition of vimentin expression as a mesenchymal marker of EMT.

Introduction

Breast cancer is the leading cause of cancer death globally. It is the second most common cause of cancer deaths among women and represents 14.3% of all deaths in the developing countries [1], and represents 38.8% of female cancers in Egypt [2]. The primary cause of breast cancer death is metastasis, which is regulated by several factors and signaling pathways, such as epithelial-mesenchymal transition (EMT), which is defined as the loss of epithelial characteristics and acquiring a mesenchymal phenotype. However, two recent studies highlighted an unexpected role of EMT in cancer drug resistance, while challenging the role of EMT in cancer metastasis [3, 4].

The mechanism by which an epithelial cell is able to acquire a mesenchymal phenotype underlies a loss of specialized epithelial cell adhesion molecules like E-cadherin, ZO-1 and cytokeratin and acquisition of mesenchymal-associated molecules like N-cadherin, vimentin and fibronectin. These changes allow the epithelial cancer cells to gain more invasiveness and metastatic capabilities [5-7].

Cadherins are a family of Ca2+-dependent cell-cell adhesion receptors that play a major role during embryonic development and in the maintenance of adult tissue architecture [8]. The cadherin family includes epithelial (E), neural (N) and placental (P) cadherins. E-cadherin, which is present in most normal epithelial cells, is related to the differentiation of epithelial cells. Loss of adhesive function of E-cadherin promotes the epithelial cells to a dedifferentiated and invasive malignant stage [9]. Clinical studies in patients with various human malignancies have also shown that E-cadherin expression is associated with dedifferentiated and lymphogenous spread of tumors [10,11]. Cell-Cell-interactions have been recognized as one of the vital regulators of apoptosis, and that the apoptotic cell death occurring during invasion may be influenced by E-cadherin-mediated cell-cell interaction between tumor cells [12].

Zonula occludens-1 (ZO-1) is a tight junction protein that is found at cell-cell adhesion membrane complexes in normal epithelial cells. Depending on the degree of cell differentiation and migration, ZO-1 shuffles during EMT from the adhesion membrane complexes to the cytoplasm and then to the nucleus [13]. During EMT, the dissolution of tight junctions is accompanied by decreased occludens and claudin expressions, along with ZO-1 diffusion from cell-cell contacts [14].

Vimentin, an intermediate filament protein, is used as a marker of mesenchymal cells to distinguish them from epithelial cells [15]. It regulates cell migration and controls recycling of endocytosed cell adhesion receptors as integrins to the plasma membrane [16]. Increased expression of vimentin is used as a EMT marker in cancer [17] and correlates with tumor growth, invasion and poor prognosis [18].

The mammalian Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway comprises four JAK domain-containing proteins, including JAK-1,-2, -3 and tyrosine kinase 2 (TYK2), as well as seven Signal Transducers and Activators of Transcription (STATs), STAT-1, STAT-2, STAT-3, STAT-4, STAT-5a, STAT-5b and STAT-6 [19]. Deregulation of JAK/STAT pathway has been involved in the promotion of oncogenic phenotypes, including tumorigenesis, invasion, metastasis, angiogenesis and anti-apoptosis [20,21]. In breast cancer, the JAK/STAT pathway has been reported to be altered through several mechanisms, including down-regulation of phosphotyrosine specific phosphatases and STAT-3 negative regulators (22,23), as well as elevation in the activating ligand IL-6 (24). Thus, JAK-2 inhibitors are being evaluated in patients with breast cancer [25].

Aloin, the major anthraquinone glycoside of the aloe juice, is characterized as C-glycoside of aloe-emodin. The empirical formula of aloin is C21H22O9, which is supported by the formulation 10?-D-glucopyranosyl-1,8-dihydroxy-3-hydroxymethyl-anthracene-9-one (26). In our previous studies, we reported on the antitumor activity of aloin against experimental murine tumors (ascites and solid Ehrlich carcinoma) [27,28], with no detrimental side effects on the host metabolism [29]. Further studies in our lab have shown the cytotoxicity of aloin against different types of human cancer cell lines, such as breast and ovarian adenocarcinoma cell lines [30-32]. We have also demonstrated that repeated treatment of the maximum tolerated dose of aloin (50mg/Kg b.w.) to normal rats shows no cardiotoxicity due to its strong antioxidant and scavenging activities for free radicals and reactive oxygen species [33], as well as its iron chelating activity [34].

We proposed this study to investigate the cytotoxic effect of aloin against breast cancer cell line (T47D), compared to an anthracycline analog, doxorubicin, which is a potent anticancer agent that is frequently used in the treatment of many malignancies, including breast cancer. Also, the design of this study was extended to elucidate the mechanistic role of some genes involved in EMT in the cytotoxic activities of aloin and doxorubicin on T47D cells progression, thus provide novel prognostic and therapeutic markers.

Materials and Methods

Chemicals

Aloin was obtained in a pure powder (MW 418.4) form from Macfarlan Smith LTD, (Edinburgh, UK). Doxorubicin hydrochloride (Adriblastina®) (MW 579.5) was provided as a lyophilized red powder (10 mg/vial) from Pharmacia S.P.A (Milan, Italy). RPMI-1640 enriched with L-glutamine and fetal bovine serum (FBS) were provided from Gibco® (Thermo Fisher Scientific, Scotland). Penicillin streptomycin mixture (1) and trypsin/EDTA (1) were purchased from Biowest® (South Africa). Phosphate-buffered saline (PBS; pH 7.2), crystal violet, dimethylsulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and methanol were provided from Sigma-Aldrich (Germany). Sterile tissue culture flasks (75 cm2), Petri dishes (58 cm2) and 96-well microtiter plates were obtained from Greiner® (USA).

The human breast cancer cell line (T47D), ER-?+, PR+ and HER2- (luminal A) was obtained from ATCC, Manassas, VA. It exhibits an aneuploid karyotype with a mode of 65 chromosomes (2-3 % of the cells have a chromosome number close to 100) Catalog No: HTB-133™). Cells were grown in RPMI-1640/L-glutamine medium enriched with 10% FBS and 1% penicillin-streptomycin mixture in a humidified 5% CO2 incubator (Thermo Scientific, USA) at 37°C. Subculturing was routinely carried out twice a week using trypsin/EDTA.

Compounds Preparation

Aloin solution (3.33% in distilled water) was freshly prepared and sterilized before use by filtration through 0.22 ?m filter (Millipore®, Merck, Germany). Five increasing aloin concentrations (20, 40, 60, 80 and 100 ?g/ml) were prepared by diluting the stock solution in complete RPMI-1640 cell culture medium. As for doxorubicin hydrochloride, 2 mg/ml solution was prepared in sterile distilled water, aliquoted and stored at -70°C. Four increasing doxorubicin concentrations (0.05, 0.1, 0.15 and 0.2 ?g/ml) were prepared by diluting the stock solution in complete RPMI-1640 cell culture medium.

Chemosensitivity Assay

Exponentially growing cells were enzymatically detached and a single tumor cell suspension in complete growth medium (150103 cells/ml) was prepared. Cells were seeded in a 96-well microtiter plate (200?l/well) and allowed to attach for 24 h in a humidified 5% CO2 incubator at 37°C. After cell attachment, the culture medium in each well was aspirated and replaced with 200?l of fresh complete growth medium containing the different aloin or doxorubicin concentrations (3 wells per each dose) and allowed to grow for 24 and 72 h, respectively. At the end of the exposure periods, 100?l of MTT (2 mg/ml in PBS) were added to each well and the plate was incubated at 37°C for further 2h. After careful aspiration of the culture medium, 150 µl DMSO were added to each well and the plate was left to stand for 1h at room temperature, and then read in an ELISA reader at 595 nm against blank (DMSO). The percentage of cell viability was calculated by multiplying the ratio absorbance of the sample versus the control by 100. Drugs IC50 were determined as aloin and doxorubicin concentrations showing 50% cell growth inhibition as compared with control cell growth [35].

Clonogenic Assay

A single tumor cell suspension in complete growth medium was prepared at a density of 6000 cells/ml. Aliquots of cell suspension (5 ml) were then transferred to 75 cm2 tissue culture flasks and incubated in a humidified CO2 incubator at 37°C for 24 h. Three culture flasks were set up for untreated cells (control) and for each compound concentration at each exposure period (24 and 72 h). After cell attachment, the culture medium was decanted and replaced with 5 ml of fresh complete growth medium in the control flasks and with 5 ml of fresh complete growth medium containing the different concentrations of aloin or doxorubicin, and then re-incubated at 37°C for 24 and 72 h, respectively. After incubation, the media were aspirated and the cells were trypsinized, collected in 15 ml falcon tubes containing fresh complete growth medium, centrifuged, and then the cell pellets were re-suspended in 4 ml of fresh complete growth medium.

The viable cell numbers were counted by trypan blue exclusion method, and then the cells were diluted with the complete growth medium to 1000 cells/ml. A volume of 1 ml cell suspension was transferred to three Petri dishes for control and for each compound concentration followed by the addition of 3 ml complete growth medium. The dishes were incubated in a humidified 5% CO2 incubator at 37°C for 10 days, during which the drug-free growth medium was replaced every 72h. At the end of the incubation period, the growth medium was decanted and the colonies were fixed in absolute methanol for 20 min, then stained with 2% crystal violet and counted using a stereomicroscope (Olympus, Japan). The number of colonies was scored by counting cell aggregates consisting of at least 50 cells (?5 generations). At least 200 tumor cell colonies per flask were required in the control Petri dish to assure an adequate range for measurement of the compound effect. The mean colony count for control was taken as 100% survival (0% inhibition) and the percent inhibition of colony formation (ICF %) in compound-treated Petri dishes was calculated [30, 36].

Quantitative Real-Time Polymerase Chain Reaction

The mRNA expression of Janus kinase-2 (JAK-2) gene and PR-target gene signal transducer and activator of transcription-5a (STAT-5a) were studied in T47D cells exposed to aloin or doxorubicin for 72h using quantitative real-time polymerase chain reaction (qRT-PCR). Also, epithelial markers including E-cadherin and zonula occludens-1 (ZO-1), and a mesenchymal marker, vimentin gene were examined. Briefly, counted T47D cells were distributed equally into three 75 cm2 tissue culture flasks containing fresh cell culture medium, incubated for 48 h in a humidified atmosphere with 5% CO2 at 37°C to allow monolayer formation. The growth medium was exchanged with a fresh one containing either no compound (control) or IC50 value of aloin or doxorubicin, and re-incubated in a humidified 5% CO2 incubator at 37°C for 72 h.

After incubation, the cell culture medium was aspirated and the cells were washed with PBS, and then trypsinized for 2 min at 37°C, followed by the addition of fresh complete growth medium. The cell suspension was centrifuged and the supernatant discarded, then the cells were immediately processed for RNA extraction. Total cellular RNA was extracted from T47D cells using RNeasy® Mini kit (Qiagen, Germany), quantified by NanoDrop One (Thermo Fisher Scientific, WI, USA), and then reversely transcribed by using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA). Real-time quantitative polymerase chain reaction (RT-PCR) amplification of the candidate genes was done using QuantiTect SYBR Green PCR Kit and QuantiTect Primer Assays (Qiagen, Germany) (Table 1) in Applied Biosystems StepOne™ Analyzer (Applied Biosystems, Waltham, Massachusetts, USA). The relative expression of selected genes was determined by the ??CT method [37].

Statistical Analysis

The Shapiro-Wilks test for normality (p0.05) showed that all data were normally distributed [38]. Statistical analysis of the difference between means was carried out using one-way analysis of variance (ANOVA). In case of a significant F-ratio, post hoc Dunnett’s and Bonferroni’s tests for multiple comparisons were used to evaluate the statistical significance between treatment groups at p2 as up-regulated and G2M). This strongly suggests that aloin does not inhibit DNA synthesis, and that cells replicate a full complement of DNA but have difficulty in mitosis [30]. Further studies in our lab have shown that treatment of human breast cancer cell lines, with (SKBR-3) and without (MCF-7) erbB-2/topoisomerase II? co-amplification, with aloin causes a decrease in the fraction of cells undergoing mitosis via induction of apoptosis, inhibition of topoisomerase II? protein expression, and down-regulation of cyclin B1 protein expression in MCF-7 cell line. In SKBR-3 cell line, erbB-2 protein expression is not affected and topoisomerase II? protein expression is mildly down-regulated at higher concentrations of aloin only [32].

In the current study, results obtained from the MTT assay of aloin and doxorubicin in the 72h exposure regimen were used to calculate their IC50 values, which were found to be 75.93µg/ml (181.5µM) and 0.099µg/ml (0.17µM), respectively (Fig.3) to be used in subsequent studies. A comparable result was previously obtained by Lee et al. [40] who reported on the cytotoxic activity of aloin against lung cancer cell line at IC50 value of 150 µM.

The foregoing findings strived the studying of the effect of continuous exposure of T47D cells to IC50 values of aloin and doxorubicin on the expression of JAK-2 and STAT-5a genes, as well as some gene markers for EMT. Despite the homology in the structure of aloin and doxorubicin, they exerted different actions on the expression levels of JAK-2 and STAT-5a mRNA. Herein we demonstrate that exposure of T47D cells to aloin (181.5µM) for 72h significantly down-regulates the relative expression levels of JAK-2 and STAT-5a mRNA, whereas doxorubicin does appear to affect them (Fig.4). Thus, aloin is recognized as a potent inhibitor of JAK-2/STAT-5a signaling pathway. The inhibition of JAK-2/STAT-5a signaling along with the sharp reduction in the percentage of cell viability provide insight into the important and regulatory role of aloin in invasive breast cancer. Recently, aloin has also been shown to inhibit JAK-1, STAT-1 and STAT-3 phosphorylation in a dose-dependent manner, and the nuclear translocation of STAT-1 and STAT-3 in lipopolysaccharide-induced inflammation in RAW 264.7 macrophages [41].

This suggests that aloin might act as a pan inhibitor of JAKS. Previously, Reiterer and Yen [42] affirmed that the inhibition of JAK-2 gene causes a significant reduction in cell growth, G2 arrest and endoreduplication in myeloblastic leukemia cells. Additionally, the inhibition of JAK-2 gene in a human colorectal cancer cell line (SW1116) with the DNA methyltransferase inhibitor, 5-aza-deoxycytidine, induced G2 cell cycle arrest and apoptosis in SW116 cells through regulation of downstream targets of JAK-2/STAT-3/STAT-5 signaling [43]. The foregoing findings correlate well with our previous studies [30, 32]. Britschgi et al. [44] reported that inhibition of JAK-2 by genetic or pharmacological means bypasses resistance to PI3K/mTOR inhibition and decreases cancer cell number, tumor growth and metastasis, as well as increasing in vivo survival, and that combined inhibition of JAK-2/STAT-5 and PI3K/mTOR leads to activation of the pro-apoptotic protein Bim and degradation of the anti-apoptotic protein Mcl-1. This explanation warrants the investigation of the effect of aloin on PI3K/AKT/mTOR.

Exploring of the effect of treating T47D cells with IC50 values of aloin on the mRNA expression of some EMT-related gene markers displayed a significant inhibition in the vimentin gene expression, whereas the expression of E-cadherin and ZO-1 genes were not changed (Fig,4). The positive correlation between increased expression of vimentin gene and tumor aggressiveness was previously documented [45, 46], and attributed to the lack of steroid receptors [16, 47]. The literature contains several studies on the regulatory effect of JAK-2 on vimentin synthesis. Colomiere et al. [48] reported that the treatment of EGF-induced OVCA433 cells with a JAK-2 inhibitor (AG490) results in the inhibition of STAT-3 activation, as well as inhibition of the expression of N-cadherin and vimentin proteins. Additionally, Stewart et al. [18] found out that a pan JAK inhibitor (JAK inhibitor I) significantly inhibited the expression levels of vimentin mRNA and protein in EGF-mediated EMT induction in MDA-MB-468 breast cancer cells. Taken altogether, it is emphasized that aloin which acts as a JAK-2/STAT-5a inhibitor reduces EMT through inhibition of the expression of vimentin mRNA.

On the other hand, doxorubicin (0.17 µM) exposure for 72 h increases the expression of E-cadherin mRNA in T47D cells, while the expression of other candidate genes remain unchanged (Fig.4). Our findings are in accordance with [49], who found out that cell-cell adhesion of YMB-S cells (which proliferate without aggregation) was increased on day 2 and day 4 after exposure to adriamycin (0.4 µM) associated with increased expression of E-cadherin and ?-catenin mRNA and protein levels. The authors reported that cell-cell adhesion induced by adriamycin is induced by increased expression of E-cadherin and decreased MUC1 expression. E-cadherin mediates early cellular adhesion events that are necessary for the formation of junctional complexes including gap junction intercellular communication (GJIC) [50], which is also important for apoptosis to occur in solid tumors [51]. If intercellular communication is required for an apoptotic signal to be transferred to cells in solid tumors, E-cadherin-mediated cell-cell adhesion may be beneficial to apoptotic signal transduction elicited by adriamycin [49].

In conclusion, although aloin and doxorubicin are anthracyclines, yet they act in a different way. Aloin is recognized as a potent inhibitor of JAK-2/STAT-5a signaling, which results in the inhibition of the expression of the mesenchymal gene marker, vimentin mRNA, that could be useful markers to explore as potential prognostic and therapeutic targets in ER+ breast cancer.

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